WO2022166918A1 - Anti-il-5 antibody formulation, preparation method therefor and use thereof - Google Patents

Abstract

Provided is an anti-IL-5 antibody formulation comprising an anti-IL-5 antibody, a His buffer, a stabilizer and Tween 80 and having a pH value of 5.70-6.38. The anti-IL-5 antibody formulation of the present invention exhibits better stability than the control under conditions of high temperatures, freezing-thawing, light, etc. being more suitable for clinical use.

Description

A kind of anti-IL-5 antibody preparation and preparation method and application thereof technical field

The invention belongs to the field of biological preparations, and relates to an anti-IL-5 (interleukin 5) antibody preparation.

Background technique

IL-5 plays an important role in many diseases and is a commonly used therapeutic target. Anti-IL-5 antibodies, such as mepolizumab, can specifically bind to IL-5, block the binding of IL-5 to its receptors, and play a role in the treatment of various diseases. Although mepolizumab has demonstrated its excellent effect, for the purpose of drug efficacy, the art still requires the preparation of anti-IL-5 antibody to be more stable.

SUMMARY OF THE INVENTION

Based on this, the present invention provides a stable anti-IL-5 antibody preparation.

In a first aspect, an anti-IL-5 antibody formulation of the invention comprises an anti-IL-5 antibody and a histidine buffer.

In one embodiment, the pH of the antibody formulation is 5.3-6.8. In one embodiment, the pH of the antibody formulation is 5.6-6.5. In one embodiment, the pH of the antibody formulation is 5.70-6.38. In one embodiment, the pH of the antibody formulation is 5.7-6.3. In one embodiment, the antibody formulation has a pH of about 5.3, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.8 , or a range (including endpoints) between any two of these values or any value therein. In one embodiment, the pH is about 5.9 or 6.0.

In one embodiment, the concentration of the histidine buffer is 10-30 mM. In one embodiment, the concentration of the histidine buffer is 19-23 mM. In one embodiment, the concentration of histidine buffer is about 10 mM, about 12 mM, about 15 mM, about 16 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 25 mM, about 27 mM , about 30 mM, or a range (including endpoints) between any two of these values or any value therein. In one embodiment, the concentration of the histidine buffer is 20 mM.

In one embodiment, the histidine buffer includes histidine and histidine hydrochloride.

In one embodiment, the antibody preparation does not include phosphoric acid or a salt thereof. In one embodiment, the antibody formulation does not include citric acid or a salt thereof.

In one embodiment, the antibody formulation further comprises stabilizers and/or surfactants.

In one embodiment, the concentration of the stabilizer is 35-130 mg/mL. In one embodiment, the stabilizer is sucrose or sorbitol. In one embodiment, the concentration of sucrose is 75-130 mg/mL. In one embodiment, the concentration of sucrose is 80-120 mg/mL. In one embodiment, the concentration of sucrose is 90-100 mg/mL. In one embodiment, the sucrose is at a concentration of about 75 mg/mL, about 78 mg/mL, about 79 mg/mL, about 80 mg/mL, about 81 mg/mL, about 82 mg/mL, about 83 mg/mL, about 88 mg/mL, about 99 mg/mL, about 105 mg/mL, about 110 mg/mL, about 117 mg/mL, about 118 mg/mL, about 119 mg/mL, about 120 mg/mL, about 121 mg/mL, about 122 mg/mL, about 123 mg/mL, about 130 mg/mL, or a range (including endpoints) between any two of these values, or any value therein.

In one embodiment, the concentration of sorbitol is 35-50 mg/mL. In one embodiment, the concentration of sorbitol is 38-42 mg/mL. In one embodiment, the concentration of sorbitol is about 35 mg/mL, about 38 mg/mL, about 39 mg/mL, about 40 mg/mL, about 41 mg/mL, about 42 mg/mL, about 45 mg/mL, about 50 mg/mL, or the range (including endpoints) between any two of these values, or any value therein.

In one embodiment, the surfactant is Tween 80 (ie, polysorbate 80) or Tween 20 (ie, polysorbate 20). In one embodiment, the concentration of surfactant is 0.1-0.4 mg/mL. In one embodiment, the concentration of surfactant is about 0.1 mg/mL, 0.15 mg/mL, 0.19 mg/mL, 0.2 mg/mL, 0.22 mg/mL, 0.24 mg/mL, 0.3 mg/mL, 0.34 mg/mL mg/mL, 0.4 mg/mL, or a range (including endpoints) between any two of these values, or any value therein. In one embodiment, the concentration of Tween 80 is 0.1-0.3 mg/mL. In one embodiment, the concentration of Tween 80 is about 0.2 mg/mL.

In one embodiment, the formulation may further comprise a chelating agent. In one embodiment, the concentration of the chelating agent is 0.01-0.03 mg/mL. In one embodiment, the concentration of the chelating agent is about 0.01 mg/mL, about 0.015 mg/mL, about 0.016 mg/mL, about 0.017 mg/mL, about 0.018 mg/mL, about 0.019 mg/mL, about 0.02 mg/mL, about 0.021 mg/mL, about 0.022 mg/mL, about 0.023 mg/mL, about 0.026 mg/mL, about 0.03 mg/mL, or a range between any two of these values ( including endpoints) or any value in it. In one embodiment, the chelating agent is EDTA (ie, ethylenediaminetetraacetic acid) or its salts, hydrates or crystals thereof, such as EDTA-2Na, EDTA disodium dihydrate. In one embodiment, the concentration of disodium EDTA (ie, EDTA-2Na) or disodium EDTA dihydrate (ie, EDTA _- 2Na.2H2O) is about 0.017-0.025 mg/mL. In one embodiment, the concentration of EDTA-2Na is about 0.017 mg/mL, about 0.018 mg/mL, about 0.019 mg/mL, about 0.02 mg/mL, about 0.021 mg/mL, about 0.022 mg/mL, about 0.023 mg/mL, about 0.025 mg/mL. In one embodiment, the concentration of disodium EDTA dihydrate is about 0.017 mg/mL, about 0.018 mg/mL, about 0.019 mg/mL, about 0.02 mg/mL, about 0.021 mg/mL, about 0.022 mg/mL mL, about 0.023 mg/mL, about 0.025 mg/mL. In one embodiment, the molar concentration of EDTA-2Na or EDTA _- 2Na.2H2O is 0.026-0.089 mM. In one embodiment, the molar concentration of EDTA-2Na or EDTA _- 2Na.2H2O is 0.026-0.080 mM. In one embodiment, the molar concentration of EDTA-2Na or EDTA-2Na·2H ₂ O is about 0.026 mM, about 0.030 mM, about 0.035 mM, about 0.041 mM, about 0.046 mM, about 0.049 mM, about 0.050 mM, about 0.051 mM, about 0.052 mM, about 0.053 mM, about 0.054 mM, about 0.063 mM, about 0.070 mM, about 0.080 mM, about 0.089 mM, or a range between any two of these values (including endpoints) or any of the values.

In one embodiment, the concentration of anti-IL-5 antibody in the formulation is 50-150 mg/mL. In one embodiment, the concentration of anti-IL-5 antibody in the formulation is 80-120 mg/mL. In one embodiment, the concentration of anti-IL-5 antibody in the formulation is about 50 mg/mL, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 94 mg/mL, about 96 mg/mL, about 98 mg/mL, about 99 mg/mL, about 100 mg/mL, about 101 mg/mL, about 102 mg/mL, about 103 mg/mL, about 104 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, about 150 mg/mL, or a range (including endpoints) between any two of these values, or any value therein.

In one embodiment, the antibody formulation comprises 50-150 mg/mL anti-IL-5 antibody, 10-30 mM histidine buffer, 80-120 mg/mL sucrose, 0.1-0.4 mg/mL Tween 80, antibody The pH of the formulation is 5.6-6.5. In one embodiment, the antibody formulation comprises 50-150 mg/mL anti-IL-5 antibody, 10-30 mM histidine buffer, 80-120 mg/mL sucrose, 0.1-0.4 mg/mL Tween 80, antibody The pH of the formulation was 5.70-6.38. In one embodiment, the antibody formulation comprises 50-150 mg/mL anti-IL-5 antibody, 10-30 mM histidine buffer, 35-50 mg/mL sorbitol, 0.1-0.4 mg/mL Tween 80, The pH of the antibody preparation was 5.70-6.38. In one embodiment, the pH of the antibody formulation is 6.0. In one embodiment, the antibody formulation comprises 50-150 mg/mL anti-IL-5 antibody, 10-30 mM histidine buffer, 80-120 mg/mL sucrose, 0.1-0.4 mg/mL Tween 80, 0.01 - 0.03 mg/mL EDTA-2Na (or 0.030-0.089 mM EDTA-2Na), pH 5.6-6.5 for antibody preparations. In one embodiment, the antibody formulation comprises 50-150 mg/mL anti-IL-5 antibody, 10-30 mM histidine buffer, 80-120 mg/mL sucrose, 0.1-0.4 mg/mL Tween 80, 0.01 - 0.03 mg/mL EDTA-2Na (or 0.030-0.089 mM EDTA-2Na) pH 5.70-6.38 for antibody preparations. In some embodiments, the formulation includes about 0.017 mg/mL EDTA-2Na (ie, about 0.051 mM EDTA-2Na). In one embodiment, the pH of the antibody formulation is 5.8-6.1.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 120 mg/mL sucrose, about 0.2 mg/mL Tween 80, about 0.017 mg/mL EDTA-2Na, pH about 6.0.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.2 mg/mL Tween 80, about 0.017 mg/mL EDTA-2Na, pH about 6.0.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.2 mg/mL Tween 80, and a pH of about 5.7- 6.4.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.2 mg/mL Tween 80, and a pH of about 5.7- 6.3.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.2 mg/mL Tween 80, and a pH of about 6.0.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.2 mg/mL Tween 80, and a pH of about 5.7.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 80 mg/mL sucrose, about 0.2 mg/mL Tween 80, and a pH of about 6.3.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 40 mg/mL sorbitol, about 0.2 mg/mL Tween 80, about 0.017 mg/mL EDTA-2Na, the pH is about 6.0.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 40 mg/mL sorbitol, about 0.2 mg/mL Tween 80, and a pH of about 6.0 .

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 120 mg/mL sucrose, about 0.2 mg/mL Tween 80, and a pH of about 6.38.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 120 mg/mL sucrose, about 0.2 mg/mL Tween 80, and a pH of about 6.0.

In one embodiment, the formulation comprises about 100 mg/mL anti-IL-5 antibody, about 20 mM histidine buffer, about 120 mg/mL sucrose, about 0.2 mg/mL Tween 80, and a pH of about 5.7. In some embodiments, the light chain of the anti-IL-5 antibody comprises a sequence as set forth in SEQ ID NO:1, or a sequence that is at least 90% identical to SEQ ID NO:1, or a sequence that is at least 90% identical to SEQ ID NO:1 : 1 compared to amino acid sequences with one or more conservative amino acid substitutions; and/or

The heavy chain of the anti-IL-5 antibody comprises the sequence shown in SEQ ID NO: 2, or a sequence with at least 90% identity compared with SEQ ID NO: 2, or a sequence with SEQ ID NO: 2 or amino acid sequence with multiple conservative amino acid substitutions. In some embodiments, the light chain of the anti-IL-5 antibody comprises the sequence set forth in SEQ ID NO:1, and the heavy chain of the anti-IL-5 antibody comprises the sequence set forth in SEQ ID NO:2. In some embodiments, the anti-IL-5 antibody is mepolizumab or a biosimilar thereof.

In some embodiments, the present invention provides a preparation method of the preparation, which includes: taking each component in a recipe amount, adding water to dissolve and mixing, and adjusting the pH value to 5.6-6.5 to obtain an antibody preparation.

In some embodiments, the present invention provides a method for preparing the formulation, comprising: preparing a histidine buffer, changing the anti-IL-5 antibody to the histidine buffer by ultrafiltration, adding excipients, and diluting the antibody to a specified level concentration to obtain antibody preparations.

In some embodiments, the present invention provides a method for preparing the formulation, comprising: preparing a 10-30 mM histidine buffer, changing the anti-IL-5 antibody to a 10-30 mM histidine buffer by ultrafiltration, Add excipients, and dilute the antibody to 80-120 mg/mL to obtain an antibody preparation.

In a second aspect, the present invention provides a method for preparing the preparation, comprising: taking a specified amount of anti-IL-5 antibody, histidine, histidine hydrochloride (or histidine hydrochloride monohydrate), sucrose or sorbitol , Tween 80, add water to dissolve and mix the above substances, adjust the pH value (such as with NaOH) to 5.6-6.5. In one embodiment, the pH is 5.70-6.38. In one embodiment, the pH is about 6.0.

In a third aspect, the present invention provides the use of the formulation in the manufacture of a product for the treatment of diseases including but not limited to asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma granulocytic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatous disease with polyangiitis, hypereosinophilic syndrome, Nasal polyposis, bullous pemphigoid, and eosinophilic esophagitis.

In a fourth aspect, the present invention provides an antibody pharmaceutical preparation for treating IL-5-related diseases, comprising the anti-IL-5 antibody preparation as described above and a container for storing the preparation. The pharmaceutical product may also include instructions for use. The container may be any container conventionally used in the art for storing pharmaceuticals, such as prefilled containers, prefilled syringes, injection pens, vials (eg, vials), ampoules, sachets, and the like. The antibody preparation of the present invention has good stability at high temperature and room temperature, can be kept in liquid form, and is convenient to use.

The preparation of the present invention shows better stability than the control under conditions of high temperature, freezing and thawing, illumination and the like, and is more suitable for clinical use.

In one embodiment, the formulations of the present invention are stable when stored at 40°C for at least 2 weeks. In one embodiment, the formulations of the invention are stable when stored at 40°C for at least 4 weeks. In one embodiment, the formulations of the present invention are stable under light conditions for at least 14 days.

In one embodiment, the formulations of the present invention are stable with at least 5 repeated freeze-thaw cycles.

specific implementation

The present invention will be described below through specific embodiments, but the content of the present invention is not limited thereto.

"About" or "approximately" refers to the conventional error range of the corresponding numerical value readily known to those skilled in the relevant art. In some embodiments, references herein to "about" or "approximately" refer to the recited value and ranges of ±10%, ±5%, or ±1% thereof.

"Comprising" or "comprising" means that the compositions and methods, etc., include the recited elements (eg, components in the compositions, steps in the methods, etc.), but do not exclude other elements. When "consisting essentially of" is used to define compositions and methods, it is meant to exclude other elements that have an essential effect on the combination for its intended use, but does not exclude elements that do not materially affect the characteristics of the composition or method . "Consisting of" means excluding elements not specifically recited. Embodiments defined by each of these transition terms are within the scope of this invention. For example, when a composition is described as including ingredients A, B, and C, compositions consisting essentially of A, B, and C and compositions consisting of A, B, and C are independently within the scope of the invention .

"Amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which can be encoded by a nucleic acid directly or in a precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides, so-called codons or base triplets. Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine Amino acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I) ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

"Conservative amino acid substitution" refers to the replacement of one amino acid residue by another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. Examples of amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.

The number of amino acids for a "conservative amino acid substitution of a heavy or light chain" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10 , about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (including endpoints) or any value therein.

"At least 90% identity" is about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 96% identity, About 97% identity, about 98% identity, about 99% identity, or a range (including endpoints) between any two of these values, or any value therein. "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions within each sequence that can be aligned. A molecule is homologous when a position in the sequences being compared is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.

The active ingredient in the formulation of the present invention is an anti-IL-5 antibody, including but not limited to monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single chain antibodies, Fab, Fab- and F(ab')2 fragments , Fv, scFvs and Fab expression libraries. In some embodiments, the anti-IL-5 antibody is a recombinant anti-IL-5 monoclonal antibody made from a cell highly expressing recombinant anti-IL-5 monoclonal antibody after cell culture, antibody isolation and high purification . In some embodiments of the present invention, the amino acid sequence of the light chain of the anti-IL-5 antibody is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO:2. In some embodiments, the anti-IL-5 antibody is mepolizumab, such as

antibodies or their biosimilars.

"Treatment" of a patient's disease refers to (1) preventing the appearance of the disease in a patient who is predisposed or not yet showing symptoms of the disease; (2) inhibits the disease or prevents its progression; or (3) reduces the disease or causes its regression.

The term "buffer" is also referred to as a buffer system or buffer system, histidine buffers include histidine and histidine salts such as hydrochloride. The amount of buffer in the present invention refers to the total amount of buffer pairs in the buffer system that constitutes the buffer. In some embodiments, molarity is employed as the unit for the amount of buffer, the value of which refers to the molarity of the buffer pair in the buffer system of the buffer. For example, in the case of a histidine buffer consisting of histidine and histidine hydrochloride, a given concentration of histidine buffer (eg, 20 mM) is the combined concentration of histidine and histidine hydrochloride.

The formulation of the present invention can be formulated with the adjuvant or its hydrate. For example, histidine hydrochloride, also known as histidine hydrochloride, can be anhydrous histidine hydrochloride or histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate; EDTA -2Na can be anhydrous EDTA-2Na or EDTA-2Na hydrate, such as EDTA-2Na·2H ₂ O. As described in "0.051mM EDTA-2Na", it can be 0.051mmol EDTA-2Na or EDTA-2Na·2H ₂ O dissolved in solvent to form 1L solution; 0.017mg EDTA-2Na, including 0.017mg EDTA-2Na Or the corresponding amount of hydrate (eg 0.019mg EDTA-2Na·2H ₂ O).

Tween is also known as polysorbate (eg, Tween 20 is known as polysorbate 20 and Tween 80 is known as polysorbate 80).

As used herein, "stability", "stable" means that in a liquid formulation comprising an antibody, the antibody (including antibody fragments thereof) does not occur, or only minimally, under the given production, preparation, transportation and/or storage conditions Aggregation, degradation or fragmentation occurs. A "stable" formulation retains biological activity under given conditions of manufacture, preparation, transportation and/or storage. The degree of aggregation, degradation or fragmentation of the formulation, etc., which can be measured by techniques such as SEC-HPLC, IEC-HPLC, CE-SDS (NR), light detection and turbidity, insoluble particles, DLS detection of particle size, etc., thereby The stability of the antibodies was assessed.

In some embodiments, the formulations provided herein have a pH of about 5.70-6.38. In some embodiments, the pH of the formulations provided herein is about 6.0.

The formulations provided by the present invention have enhanced stability. In some embodiments, the formulations of the present invention are stable over at least 5 freeze-thaw cycles. In other embodiments, the formulation of the present invention remains stable after being placed at a high temperature of 40°C for 1 week or 2 weeks. In other embodiments, the formulation of the present invention can remain stable for a period of time under 4000lx light conditions.

The present invention also provides a method of treating an IL-5-related disease, the method comprising administering the antibody preparation to a patient.

In the following examples, unless otherwise specified, the reagents and instruments used are conventional reagents and instruments in the field, which can be obtained commercially; the methods used are conventional technical methods in the art, and those skilled in the art can refer to the examples The content of the method can be implemented without question and the corresponding results can be obtained.

The anti-IL-5 antibody used in the following examples was Mepolizumab. Mepolizumab contains 2 light chains with the same sequence and 2 heavy chains with the same sequence, the amino acid sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2 antibodies respectively:

SEQ ID NO: 1 (light chain sequence):

SEQ ID NO: 2 (heavy chain sequence):

The anti-IL-5 antibodies used in the following examples were expressed from CHO cells, produced after cell culture, isolation and high purification. CHO cells, antibody expression methods, plasmid construction and purification methods were purchased and operated in conventional manner.

In the following examples, SEC-HPLC, IEC-HPLC and osmotic pressure detection were carried out in a conventional manner.

Example 1: Effects of different pH values on formulation stability

Prepare 20mM histidine buffer with pH 7.7 (weigh 3.1005g histidine, dissolve in 1L water and mix) and 20mM histidine buffer with pH 4.1 (weigh 4.2032g histidine hydrochloride) , dissolved in 1 L of water, and mixed), and then intermodulate the above two buffers into 20 mM histidine buffer with pH 5.2 and pH 6.5. The purified anti-IL-5 antibody was ultrafiltered into buffers of pH 5.2 and pH 6.5, respectively, and the two pH samples obtained were intermodulated. Sucrose and Tween 80 were added according to the prescription ingredients in Table 1, and then the antibody Dilute to 100 mg/mL, and finally obtain samples with pH 6.77, 6.38, 6.00, 5.70, and 5.34 with antibody concentration of 100 mg/mL, respectively.

The other ingredients and contents of each formulation are shown in Table 1 below:

Table 1: Formulation formulations for different pH values

prescription number	4	5	6	7	8
His (histidine buffer) (mM)	20	20	20	20	20
Sucrose (mg/ml)	120	120	120	120	120
Tween 80 (mg/ml)	0.2	0.2	0.2	0.2	0.2
pH	6.77	6.38	6.00	5.70	5.34

experiment method:

The prepared and subpackaged samples were placed at 40°C, and samples were taken at 0 days, 3 days, 7 days, and 14 days for detection. The detection items were: SEC-HPLC and IEC-HPLC. The present embodiment studies the effects of the same composition and different pH values on the stability of the preparation, and the test results are as follows:

Table 2: SEC-HPLC data of different formulations at 40°C

It can be seen from Table 2 that in the range of pH value of 5.34 to 6.77, the monomer purity of each preparation is kept well, and the SEC monomer purity of prescription 5 to prescription 8 (pH5.34-pH6.38) is lower than that of prescription 4. (pH6.77) less.

Table 3: IEC-HPLC data of different formulations at 40°C

From the results in Table 3, it can be seen that the main peak content of each preparation is well maintained within the pH range of 5.34 to 6.77. The IEC main peak content of prescription 5 to prescription 7 (pH5.70-pH6.38) is higher than that of prescription 8 (pH5.38). 34) and formulation 4 (pH6.77) showed a slower decline in the IEC main peak content.

From the above results, it can be seen that under the same ingredients, in the range of pH 5.70-pH 6.38, the stability of each formulation is better.

Example 2: Effects of different buffers on formulation stability

The effect of different buffers on formulation stability was analyzed. Control formulation 3 (ingredients: 100mg/ml anti-IL-5 antibody, 4.5mM citric acid monohydrate, 15.5mM disodium hydrogen phosphate heptahydrate, 120mg/ml sucrose, 0.2mg/ml Tween 80, pH 6.36) Unlike prescription 5, only the buffer solution, other components, contents and pH values are basically the same. Comparing the two prescriptions, the results are as follows:

Table 4: SEC-HPLC data under different buffer conditions

It can be seen from Table 4 that the SEC-HPLC monomer content of recipe 5 decreased more slowly than the monomer content of recipe 3, indicating that the buffer of recipe 5 was more able to keep the formulation stable.

Table 5: IEC-HPLC data for different buffers at 40°C

As can be seen from Table 5, the IEC-HPLC main peak content of prescription 5 decreased more slowly than the main peak content of prescription 3. Combined with the data in Table 4 above, it can be seen that different buffers have a great influence on the stability of the preparation, among which, His buffer is better than the buffer composed of citric acid and disodium hydrogen phosphate.

Based on the above experimental results: for the anti-IL-5 antibody preparation, when the pH range is 5.70-6.38, and the buffer is His buffer, the stability of the preparation is stronger.

Example 3: Formulation Stability Study

This example investigates the stability of several formulations and compares them to controls.

Among them, the preparation method of 20mM His buffer (ie 20mM histidine buffer) is as follows: weigh 99.216g of histidine and 201.752g of histidine hydrochloride, dissolve in 80L ultrapure water, mix well, pH value is 5.8. The anti-IL-5 antibody was ultrafiltered and replaced with 20 mM His buffer, and the rest of the auxiliary materials were added according to the prescription ingredients described in Table 6, and then the antibody was diluted to 100 mg/mL to obtain the samples of prescription 6, 14-18.

Table 6: Formulation samples with different ingredients

prescription number	6	12 (control)	14	15	16	17	18
His buffer (mM)	20		20	20	20	20	20
Citric acid monohydrate (mM)		4.5
Anhydrous disodium hydrogen phosphate (mM)		15.5
EDTA-2Na dihydrate (mg/mL)		0.019	0.019	0.019		0.019
Sucrose (mg/mL)	120	120	120	80	80
Sorbitol (mg/ml)						40	40
Tween 80 (mg/ml)	0.2	0.2	0.2	0.2	0.2	0.2	0.2
pH	5.95	6.25	5.97	5.98	5.95	5.97	5.98

experiment method:

The prepared and subpackaged samples were placed under the conditions of repeated freezing and thawing at 40°C, light and -60°C, respectively, and sampling was performed after a certain period of time. The placement conditions and sampling time points are as follows in Table 7:

Table 7: Sample placement conditions and sampling time

condition	Sampling time
40℃	0 days, 1 week, 2 weeks, 3 weeks, 4 weeks
illumination	0 days, 1 day, 3 days, 7 days, 14 days
-60℃ repeated freezing and thawing	0 times (DR0), 3 times (DR3), 5 times (DR5)

The inspection items are as follows:

0-day detection: osmotic pressure, SEC-HPL, IEC-HPLC;

Other time points: SEC-HPLC, IEC-HPLC (point selection).

test results

1. Osmotic pressure test results

The osmotic pressure of each formulation was detected on day 0, and the results are shown in Table 8.

Table 8: Osmolality data for each formulation sample

It can be seen from Table 8 that the osmotic pressure of prescriptions 15-18 is close to the physiological range, which is more suitable for subcutaneous injection.

2. High temperature screening results

The samples placed at 40°C were subjected to SEC-HPLC and IEC-HPLC detection, and the results are shown in Table 9 and Table 10, respectively.

Table 9: SEC-HPLC data of each formulation sample at high temperature (40°C)

As can be seen from Table 9, the SEC-HPLC monomer purity of the control formulation 12 decreased the fastest. Prescriptions 6 and 14-18 can maintain good stability, which is more stable than the control prescription, especially prescriptions 6, 14-16.

Table 10: IEC-HPLC data at high temperature (40°C)

From the results in Table 10, it can be seen that the IEC main peak content of prescriptions 6 and 14-18 decreased less than the control prescription 12, especially the IEC main peak content of prescription 14 and prescription 16 decreased the slowest.

3. Light screening results

Perform SEC-HPLC and IEC-HPLC detection on each sample placed under light conditions (25°C, 4000±500lx), and the results are as follows:

Table 11: SEC-HPLC data of each formulation sample under light conditions

It can be seen from Table 11 that the SEC-HPLC monomer purity of the control formulation 12 decreased the fastest, which was significantly higher than that of other formulations.

Table 12: IEC-HPLC data of each recipe sample under light conditions

From Table 12, it can be seen that the decrease of IEC main peak content of prescriptions 14-18 is lower than that of control prescription 12.

4. Freeze-thaw screening results

Freeze-thaw is to freeze and thaw each sample solution at -60°C for 24h, and then place it at 25°C for 24h as one freeze-thaw. The samples were tested after 0, 3, and 5 freeze-thaw times. The results are as follows: Show:

Table 13: SEC-HPLC data under repeated freeze-thaw conditions

It can be seen from Table 13 that after 5 freeze-thaw cycles, the SEC monomer purity of recipe 12 has a downward trend, and the freeze-thaw results of other recipes have no significant difference.

Combined with the experimental results of high temperature, light and freezing and thawing, formulations 6 and 14-18 have good stability compared to the control formulation 12.

Claims (40)

1. An anti-IL-5 antibody preparation, comprising an anti-IL-5 antibody and a histidine buffer.
2. The formulation of claim 1, wherein the pH of the antibody formulation is 5.6-6.5. 3. The formulation of claim 1, wherein the pH of the antibody formulation is 5.7-6.4.
3. The formulation of claim 1, wherein the pH of the antibody formulation is 5.9, 6.0, or 6.1.
4. The formulation of claim 1, wherein the concentration of the histidine buffer is 10-30 mM.
5. The formulation of claim 4, wherein the concentration of the histidine buffer is 20 mM.
6. The formulation of any one of claims 1-6, wherein said comprises a stabilizer and/or a surfactant.
7. The formulation of claim 7, wherein the stabilizer is sucrose or sorbitol.
8. The formulation of claim 7, wherein the concentration of the stabilizer is 35-130 mg/mL.
9. The formulation of claim 8, wherein the concentration of sucrose is 75-130 mg/mL, or the concentration of sorbitol is 35-50 mg/mL.
10. The formulation of claim 8, wherein the concentration of sucrose is 80 mg/mL or 120 mg/mL, or the concentration of sorbitol is 40 mg/mL.
11. The formulation of claim 7, wherein the surfactant is Tween 80 or Tween 20.
12. The formulation of claim 12, wherein the concentration of the Tween 80 is 0.1-0.4 mg/mL.
13. The formulation of claim 12, wherein the concentration of Tween 80 is 0.2 mg/mL.
14. The formulation of any one of claims 1-14, wherein the concentration of the anti-IL-5 antibody is 80-120 mg/mL.
15. The formulation of claim 15, wherein the concentration of the anti-IL-5 antibody is 100 mg/mL.
16. The formulation of any one of claims 1-16, wherein the formulation may further comprise a chelating agent.
17. The formulation of claim 17, wherein the chelating agent is EDTA-2Na.
18. The formulation of claim 17, wherein the concentration of the chelating agent is 0.01-0.03 mg/mL.
19. The formulation of claim 17, wherein the concentration of EDTA-2Na is 0.017 mg/mL.
20. An anti-IL-5 antibody preparation, wherein the preparation comprises 50-150mg/mL anti-IL-5 antibody, 10-30mM histidine buffer, 80-120mg/mL sucrose, 0.1-0.4mg/mL tween 80, the pH of the antibody preparation is 5.6-6.5.
21. An anti-IL-5 antibody preparation, wherein the preparation comprises 50-150mg/mL anti-IL-5 antibody, 10-30mM histidine buffer, 35-50mg/mL sorbitol, 0.1-0.4mg/mL titer The pH of the antibody preparation was 5.6-6.5 at temperature 80.
22. An anti-IL-5 antibody preparation, wherein the preparation comprises 50-150mg/mL anti-IL-5 antibody, 10-30mM histidine buffer, 80-120mg/mL sucrose, 0.1-0.4mg/mL tween 80, 0.01-0.03 mg/mL of EDTA-2Na, the pH of the antibody preparation is 5.6-6.5.
23. An anti-IL-5 antibody preparation, wherein the preparation comprises 100mg/mL anti-IL-5 antibody, 20mM histidine buffer, 120mg/mL sucrose, 0.2mg/mL Tween 80, 0.017mg/mL EDTA -2Na, pH about 5.6-6.5.
24. An anti-IL-5 antibody preparation, wherein the preparation comprises 100mg/mL anti-IL-5 antibody, 20mM histidine buffer, 120mg/mL sucrose, 0.2mg/mL Tween 80, 0.051mM EDTA-2Na , pH is about 5.6-6.5.
25. An anti-IL-5 antibody preparation, wherein the preparation comprises 100mg/mL anti-IL-5 antibody, 20mM histidine buffer, 80mg/mL sucrose, 0.2mg/mL Tween 80, 0.017mg/mL EDTA -2Na, pH about 5.6-6.5.
26. An anti-IL-5 antibody preparation, wherein the preparation comprises 100mg/mL anti-IL-5 antibody, 20mM histidine buffer, 80mg/mL sucrose, 0.2mg/mL Tween 80, 0.051mM EDTA-2Na , pH is about 5.6-6.5.
27. An anti-IL-5 antibody preparation, wherein the preparation comprises 100mg/mL anti-IL-5 antibody, 20mM histidine buffer, 80mg/mL sucrose, 0.2mg/mL Tween 80, pH is about 5.6-6.5 .
28. An anti-IL-5 antibody preparation, wherein the preparation comprises 100mg/mL anti-IL-5 antibody, 20mM histidine buffer, 40mg/mL sorbitol, 0.2mg/mL Tween 80, 0.017mg/mL EDTA-2Na, pH about 5.6-6.5.
29. An anti-IL-5 antibody preparation, wherein the preparation comprises 100 mg/mL anti-IL-5 antibody, 20 mM histidine buffer, 40 mg/mL sorbitol, 0.2 mg/mL Tween 80, 0.051 mM EDTA- 2Na, pH about 5.6-6.5.
30. A kind of anti-IL-5 antibody preparation, wherein, described preparation comprises 100mg/mL anti-IL-5 antibody, 20mM histidine buffer, 40mg/mL sorbitol, 0.2mg/mL Tween 80, pH is about 5.6- 6.5.
31. An anti-IL-5 antibody preparation, wherein the preparation comprises 100 mg/mL anti-IL-5 antibody, 20 mM histidine buffer, 120 mg/mL sucrose, 0.2 mg/mL Tween 80, pH 5.6-6.5 .
32. The formulation according to any one of claims 24 to 32, wherein the pH of the formulation is 5.7-6.4.
33. The formulation according to any one of claims 24 to 32, wherein the pH of the formulation is 5.9-6.1.
34. The preparation according to any one of claims 1 to 34, wherein the light chain of the anti-IL-5 antibody comprises the sequence shown in SEQ ID NO:1, or has at least 90% compared to SEQ ID NO:1 A sequence of identity, or an amino acid sequence with one or more conservative amino acid substitutions compared to SEQ ID NO: 1; and/or

    The heavy chain of the anti-IL-5 antibody comprises the sequence shown in SEQ ID NO:2, or a sequence with at least 90% identity compared with SEQ ID NO:2, or compared with SEQ ID NO:2 An amino acid sequence with one or more conservative amino acid substitutions.
35. The preparation of claim 35, wherein the light chain amino acid sequence of the anti-IL-5 antibody is shown in SEQ ID NO: 1, and the heavy chain amino acid sequence is shown in SEQ ID NO: 2.
36. The preparation according to any one of claims 1 to 34, wherein the anti-IL-5 antibody is mepolizumab.
37. The formulation of any one of claims 1-36, wherein the formulation is stable at 40°C for at least 2 weeks or at least 4 weeks; alternatively, the formulation is stored under light for at least 14 days Stable; alternatively, the formulation is stable with at least 5 freeze-thaw cycles.
38. The preparation method of the preparation of any one of claims 1-38, comprising: taking each component of the recipe, adding water to dissolve and mixing, and adjusting the pH value to 5.6-6.5 to obtain an antibody preparation;

    Alternatively, it includes: preparing a histidine buffer, changing the anti-IL-5 antibody into the histidine buffer by ultrafiltration, adding auxiliary materials, and diluting the antibody to a specified concentration to obtain an antibody preparation.
39. Use of the formulation of any one of claims 1-38 in the manufacture of a product for the treatment of a disease selected from the group consisting of asthma, severe eosinophilic asthma, severe asthma, uncontrolled eosinophilic asthma, eosinophilic asthma, sub-eosinophilic asthma, chronic obstructive pulmonary disease, eosinophilic granulomatous disease with polyangiitis, hypereosinophilic syndrome, nasal polyps disease, bullous pemphigoid, and eosinophilic esophagitis.
40. An antibody pharmaceutical preparation for treating IL-5-related diseases, comprising the preparation of any one of claims 1-38 and a container for storing the preparation.